Explorating the mechanism of Epimedii folium-Rhizoma drynariae herbal pair promoted bone defects healing through network pharmacology and experimental studies
Explorating the mechanism of Epimedii folium-Rhizoma drynariae herbal pair promoted bone defects healing through network pharmacology and experimental studies
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DOI：
10.1016/j.jep.2023.117329
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发表时间：
2023-11-01
影响因子：
5.4
通讯作者：
Shou，Dan
中科院分区：
文献类型：
--
作者：
Lei，Shan Shan;Huang，Xiao Wen;Shou，Dan
骨缺损很难治疗，骨不连的发生率很高。淫羊藿叶骨碎补对(EDP)是一种用于治疗骨科疾病的中药。然而，EDP促进成骨或骨形成的机制仍很不清楚。研究目的：本研究旨在通过网络药理学和实验相结合的方法，探讨EDP促进骨缺损的骨形成的机制。利用分子对接或网络药理学进行HUB靶标和途径富集化分析。采用大鼠骨缺损模型，通过μ、CT和组织病理学检查确定其药理作用。RT-PCR法检测EDP对Bmp2、Smad2/5、Runx2和Alp基因表达的影响；免疫组织化学和Western印迹法检测EDP对大鼠胫骨组织BMP2、COL1A1、SPP1、ALP和RUNX2蛋白表达的影响。为了更好地了解EDP对骨髓间充质干细胞(BMSCs)成骨分化的影响，我们还进行了细胞活性测定、茜素红和碱性磷酸酶染色以及RT-PCR。结果鉴定了EDP的14个关键化合物和47个HUB靶点，它们可能参与促进成骨以修复骨缺损。BMP/Smad/Runx2通路可能是EDP促进骨缺损修复的关键途径。体内实验结果表明，EDP能有效促进模型大鼠胫骨修复，激活BMP/Smad/Runx2途径，上调Bmp2、Bmpr1α、Smad2/5、Runx2和Alp基因，增加BMP2、COL1A1、Runx2和ALP的蛋白表达。体外实验发现，EDP可促进BMSCs的增殖、分化和矿化，并上调BMP/Smad/Runx2途径中关键基因的表达。
Ethnopharmacological relevanceBone defects are difficult to treat and have a high incidence of nonunion. TheEpimedii folium-Rhizoma drynariaeherbal pair (EDP) is a traditional Chinese medicine (TCM) used for treating bone diseases. However, the mechanisms by which EDP promotes osteogenesis or bone formation remain largely unclear.Aim of the study: This study aimed to investigate the mechanism of EDP promoted bone formation in bone defects using network pharmacology and experiments.Materials and methodsThe chemical components of EDP were analyzed by UHPLC-MS. The hub target and pathway enrichment analysis was conducted using molecular docking or network pharmacology. The pharmacological actions of EDP were determined by μCT and histopathology examination using a bone defect rat model. The effects of EDP on the mRNA expression ofBmp2,Smad2/5,Runx2, andAlpgenes were measured by RT-PCR, while changes in the protein expressions of BMP2, COL1A1, SPP1, ALP, and RUNX2in the tibia tissues of the rats in response to EDP were analyzed by immunohistochemical staining or Western blot. We also performed cell viability assays, Alizarin Red and ALP staining assays, and RT-PCR to better understand how EDP affected osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).ResultsIdentified 14 key compounds and 47 hub targets of EDP that may be involved in promoting osteogenesis to repair bone defects. And the BMP/Smad/Runx2 pathway was likely the key pathway through which EDP promoted bone defects repairing. The results ofin vivorat experiments indicated that EDP effectively promoted tibia repair in the model rats and activated the BMP/Smad/Runx2 pathway in the tibia tissue, with upregulatingBmp2,Bmpr1α,Smad2/5,Runx2, andAlpgenes, and increased the protein expression of BMP2, COL1A1, RUNX2, and ALP.In vitro, EDP was found to increase the proliferation, differentiation, and mineralization in BMSCs- and also up-regulated the expression of key genes in the BMP/Smad/Runx2 pathway.ConclusionThis study highlighted the ability of EDP to promote the osteogenic differentiation to enable bone repair by activating the BMP/Smad/Runx2 pathway.